Development of Novel Monoclonal Antibodies Against CD109 Overexpressed in Human Pancreatic Cancer

www.oncotarget.com Oncotarget, 2018, Vol. 9, (No. 28), pp: 19994-20007 Research Paper Development of novel monoclonal antibodies against CD109 overexpressed in human pancreatic cancer Gustavo A. Arias-Pinilla1, Angus G. Dalgleish2, Satvinder Mudan3, Izhar Bagwan4, Anthony J. Walker1 and Helmout Modjtahedi1 1School of Life Sciences, Pharmacy and Chemistry, Kingston University London, Kingston-upon-Thames, Surrey, UK 2Department of Cellular and Molecular Medicine, St George’s University of London, London, UK 3Department of Surgery of Hammersmith Campus, Imperial College, London, UK 4Department of Histopathology, Royal Surrey County Hospital, Guildford, UK Correspondence to: Helmout Modjtahedi, email: [email protected] Keywords: pancreatic cancer; monoclonal antibodies; CD109 antigen; tissue arrays; immunohistochemistry Received: December 10, 2017 Accepted: March 15, 2018 Published: April 13, 2018 Copyright: Arias-Pinilla et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Pancreatic cancer is one of the most aggressive and lethal types of cancer, and more effective therapeutic agents are urgently needed. Overexpressed cell surface antigens are ideal targets for therapy with monoclonal antibody (mAb)-based drugs, but none have been approved for the treatment of pancreatic cancer. Here, we report development of two novel mouse mAbs, KU42.33C and KU43.13A, against the human pancreatic cancer cell line BxPC-3. Using ELISA, flow cytometry, competitive assay and immunoprecipitation followed by mass spectrometry, we discovered that these two mAbs target two distinct epitopes on the external domain of CD109 that are overexpressed by varying amounts in human pancreatic cancer cell lines. Treatment with these two naked antibodies alone did not affect tumour cell growth or migration in vitro. Of the two mAbs, only KU42.33C was useful in determining the expression of CD109 in tumour cells by Western blot and immunohistochemistry. Interestingly, immunohistochemistry of human pancreatic carcinoma tissue arrays with mAb KU42.33C showed that 94% of the 65 human pancreatic adenocarcinoma cases were CD109 positive, with no expression in normal pancreatic tissues. Our results suggest that these two novel mAbs are excellent tools for determining the expression level of CD109 in the tumour specimens and sera of patients with a wide range of cancers, in particular pancreatic cancer, and for investigating its diagnostic, prognostic and predictive value. Further research is warranted and should aim to unravel the therapeutic potential of the humanised forms or conjugated versions of such antibodies in patients whose tumours overexpress CD109 antigen. INTRODUCTION Surgery is indicated in patients with localised disease, accounting only for 10-20% of cases, and is followed Pancreatic cancer is one of the most aggressive by adjuvant chemotherapy [2]. Treatment of metastatic and lethal types of cancer with a five-year survival rate disease is based on gemcitabine-containing regimens, of ~5% which has barely improved in the past four sometimes in combination with erlotinib or more decades [1]. In 2012, there were an estimated 337,872 recently with nab-paclitaxel although such combinations new cases and 330,391 deaths as a result of pancreatic only offer modest survival benefit in some patients in cancer worldwide [1]. Treatment of pancreatic cancer comparison with gemcitabine alone [3–5]. Treatment with requires a multidisciplinary approach that includes gemcitabine-free FOLFIRINOX (leucovorin, fluorouracil, surgery, chemotherapy, radiotherapy and palliative care. irinotecan, oxaliplatin) has improved the median overall www.oncotarget.com 19994 Oncotarget survival compared with gemcitabine alone (11.1 vs 6.8 KU42.33C and KU43.13A was found to be trypsin- months), but this combinational therapy is accompanied sensitive and there was no binding when screened by by increased side effects and thus is often an option for flow cytometry (data not shown). Therefore, an enzyme- younger patients with good performance status [6]. There free cell dissociation buffer was used for cell detachment is currently no reliable biomarker or screening method prior to flow cytometry analysis. Following optimisation, for the early detection of pancreatic cancer and novel and high level of expression of the target antigen was found more effective therapeutics are urgently needed. Moreover, with both mAbs KU42.33C and KU43.13A in some of without further advances in these fronts, pancreatic cancer the human pancreatic cancer cell lines including BxPC- is projected to bypass breast, prostate and colorectal 3 (MFI=86 and 90), Capan-2 (MFI=19 and 16), MIA cancers, and become the second leading cause of cancer PaCa-2 (MFI=56 and 57), PANC-1 (MFI=45 and 56), deaths after lung cancer in the USA by 2030, and the third CFPAC-1 (MFI=21 and 19) and FA-6 (MFI=39 and 36, leading cause of cancer death in European Union after respectively), compared to negative control (Figure 1, lung and colorectal cancer by 2025 [7, 8]. Supplementary Figure 2). In contrast, the expression of Monoclonal antibody (mAb) technology is the target antigen in other human pancreatic cancer cell an excellent tool for the identification of novel and lines namely Capan-1, AsPC-1, HPAF-II and Hs766T was overexpressed cell surface antigens in human malignancies found to be negative or lower with MFI values ranging and mAb-based products have untapped therapeutic from 2-12 (Figure 1, Supplementary Figure 2). We also and diagnostic potential in cancer. Indeed, the exquisite examined the level of expression of the target antigen in specificity of mAbs for their target antigens makes them other cancer cell lines including human head and neck an attractive approach for targeted cancer therapy [9–14]. squamous carcinoma HN5 and breast cancer cell lines To date, 32 mAbs have been approved for cancer treatment MDA-MB-468 and SKBR-3. High level of the target in the U.S. and/or European Union. However, none have antigen was present in both HN5 and the triple negative yet been approved for pancreatic cancer [15–18]. Our aim breast cancer cell line MDA-MB-468, but not in the HER- was to develop novel mAbs of diagnostic and therapeutic 2 overexpressing breast cancer cell line SKBR-3 (Figure potential against overexpressed cell surface antigens 1, Supplementary Figure 2). on human pancreatic cancer cells. Here, we report the Both novel mAbs were purified by affinity development of two novel mouse mAbs against distinct chromatography and were found to be of IgG1κ isotype epitopes on the extracellular domain of CD109, using the (Supplementary Figure 3). To determine whether the two human pancreatic cancer cell line BxPC-3 as the source of antibodies were directed against the same, overlapping or tumour immunogen. We showed that the CD109 antigen distinct epitopes on the target antigen, competitive ELISA is overexpressed in several human pancreatic cancer cell was performed. For this, saturation of the antigen binding lines and pancreatic cancer tissue array, with no expression site was initially performed with one the mAbs prior to in normal pancreatic tissues. The characterisation of these addition of the second antibody to explore if absorbance antibodies and their potential for use as diagnostic and value increased further. The absorbance curve for mAb therapeutic agents are discussed. KU42.33C plateaued at concentrations higher than 50 μg/ ml. Thereafter, only additional doses of mAb KU43.13A RESULTS increased the absorbance (Figure 2). These results support that mAbs KU42.33C and KU43.13A recognise non- Development of two novel mouse mAbs overlapping epitopes on the extracellular domain on the KU42.33C and KU43.13A target antigen. Several hybridoma were generated by the fusion of The two novel mAbs KU42.33C and KU43.13A lymphocytes, isolated from the spleens of mice immunised are against CD109 on human pancreatic cancer with BxPC-3 cells, and the myeloma cell line SP2. The cells hybridomas’ supernatants were screened against a panel of human pancreatic cancer cell lines established from Both mAbs, KU42.33C and KU43.13A, patients at different stages of their disease including immunoprecipitated a ~170 KDa protein identified as primary tumours (BxPC-3, Capan-2, MIA PaCa-2, PANC- CD109 by mass spectrometry (Figure 3A and Table 1), liver metastatic tumours (CFPAC-1, Capan-1), ascites 1). In addition, mAb KU42.33C immunoprecipitated (HPAF-II, AsPC-1) and lymph node metastasis (Hs 766T). faint bands of ~150 KDa and ~80 KDa (Figure 3A). The results of ELISA screening showed that the antibodies However, in Western blot, only KU42.33C detected the secreted by two hybridomas, KU42.33C and KU43.13A, CD109 bands of 170 KDa and 150 KDa, suggesting that were against an antigen with high level of expression in KU43.13A is directed against a conformational epitope some of the human pancreatic cell lines (e.g. BxPC-3, on CD109 antigen (Figures 3B). Furthermore, to confirm PANC-1, FA-6 and MIA PaCa-2, Supplementary Figure that these two antibodies were directed against CD109, 1). However, the target antigen recognised by mAbs BxPC-3 tumour lysates were immunoprecipitated with www.oncotarget.com 19995 Oncotarget the two novel antibodies, transferred by Western blot and 4A & 4B). The anti-EGFR mAb HM43.16B was used as then

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